Carboxypeptidase of cocoa

ABSTRACT

The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase and polypeptide in the manufacture of cocoa flavor and/or chocolate.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of the US nationalphase designation of International application PCT/EP02/07162 filed Jun.28, 2002, the content of which is expressly incorporated herein byreference thereto.

BACKGROUND ART

[0002] The present invention relates to a novel carboxypeptidase geneand the polypeptide encoded thereby. In particular, the presentinvention relates to the use of the present carboxypeptidase in themanufacture of cocoa flavor and/or chocolate.

[0003] It is known that in processing cacao beans the generation of thetypical cocoa flavor requires two steps—a fermentation step, whichincludes air-drying of the fermented material, and a roasting step.

[0004] During fermentation two major activities may be observed. First,the pulp surrounding the beans is degraded by micro-organisms with thesugars contained in the pulp being largely transformed to acids,especially acetic acid (Quesnel et al., J. Sci. Food. Agric. 16 (1965),441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). Theacids then slowly diffuse into the beans and eventually cause anacidification of the cellular material. Second, fermentation alsoresults in a release of peptides exhibiting differing sizes and ageneration of a high level of hydrophobic free amino acids. This latterfinding led to the hypothesis that proteolysis occurring during thefermentation step is not due to a random protein hydrolysis but seems tobe rather based on the activity of specific endoproteinases (Kirchhoffet al., Food Chem 31 (1989), 295-311). This specific mixture of peptidesand hydrophobic amino acids is deemed to represent cocoa-specific flavorprecursors.

[0005] Until now several proteolytic enzyme activities have beeninvestigated in cacao beans and studied for their putative role in thegeneration of cocoa flavor precursors during fermentation.

[0006] An aspartic endoproteinase activity, which is optimal at a verylow pH (pH 3.5) and inhibited by pepstatin A has been identified. Apolypeptide described to have this activity has been isolated and isdescribed to consist of two peptides (29 and 13 kDa) which are deemed tobe derived by self-digestion from a 42 kDa pro-peptide (Voigt et al., J.Plant Physiol. 145 (1995), 299-307). The enzyme cleaves proteinsubstrates between hydrophobic amino acid residues to produceoligopeptides with hydrophobic amino acid residues at the ends (Voigt etal., Food Chem. 49 (1994), 173-180). The enzyme accumulates with thevicilin-class (7S) globulin during bean ripening. Its activity remainsconstant during the first days of germination and does not decreasebefore the onset of globulin degradation (Voigt et al., J. PlantPhysiol. 145 (1995), 299-307).

[0007] Also a cysteine endoproteinase activity had been isolated whichis optimal at a pH of about 5. This enzymatic activity is believed notto split native storage proteins in ungerminated seeds. Cysteineendoproteinase activity increases during the germination process whendegradation of globular storage protein occurs. To date, no significantrole for this enzyme in the generation of cocoa flavor has been reported(Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, Nov.17-23, 1996).

[0008] Moreover, a carboxypeptidase activity has been identified whichis inhibited by PMSF and thus belongs to the class of serine proteases.It is stable over a broad pH range with a maximum activity at pH 5.8.This enzyme does not degrade native proteins but preferentially splitshydrophobic amino acids from the carboxy-terminus of peptides (Bytof etal., Food Chem. 54 (1995), 15-21).

[0009] During the second step of cocoa flavor production—the roastingstep—the oligopeptides and amino acids generated at the stage offermentation are obviously subjected to a Maillard reaction withreducing sugars present in fermented beans eventually yieldingsubstances responsible for the cocoa flavor as such.

[0010] In the art there have been many attempts to artificially producecocoa flavor.

[0011] Cocoa-specific aroma has been obtained in experiments whereinacetone dry powder (AcDP) prepared from unfermented ripe cacao beans wassubjected to autolysis at a pH of 5.2 followed by roasting in thepresence of reducing sugars. It was conceived that under theseconditions preferentially free hydrophobic amino acids and hydrophilicpeptides should be generated and the peptide pattern thus obtained wasfound to be similar to that of extracts from fermented cacao beans. Ananalysis of free amino acids revealed that Leu, Ala, Phe and Val werethe predominant amino acids liberated in fermented beans or autolysis(Voigt et al., Food Chem. 49 (1994), 173-180). In contrast to thesefindings, no cocoa-specific aroma could be detected when AcDP wassubjected to autolysis at a pH of as low as 3.5 (optimum pH for theaspartic endoproteinase). Only few free amino acids were found to bereleased but a large number of hydrophobic peptides were formed. Whenpeptides obtained after the autolysis of AcDP at a pH of 3.5 weretreated with carboxypeptidase A from porcine pancreas at pH 7.5,hydrophobic amino acids were preferentially released. The pattern offree amino acids and peptides was similar to that found in fermentedcacao beans and to the proteolysis products obtained by autolysis ofAcDP at pH 5.2. After roasting of the amino acids and peptides mixtureas above, a cocoa aroma could be generated.

[0012] It has also been shown that, a synthetic mixture of free aminoacids alone with a similar composition to that of the spectrum found infermented beans, was incapable of generating cocoa aroma after roasting,indicating that both the peptides and the amino acids are important forthis purpose (Voigt et al., Food Chem. 49 (1994), 173-180.

[0013] In view of the above data a hypothetical model for thegeneration, during fermentation, of the said mixture of peptides andamino acids, i.e. the cocoa flavor precursors, had been devised (FIG.1), where in a first step peptides having a hydrophobic amino acid attheir end, are formed from storage proteins, which peptides are thenfurther degraded to smaller peptides and free amino acids. To producethe said peptides having C-terminal hydrophobic amino acids, an asparticendoproteinase activity related to that mentioned above seems to beinvolved. Yet, for splitting off hydrophobic amino acids from peptidesformed in the preceding step the only known enzymatic activity, whichmight be considered in this respect, is that of a carboxypeptidase.However, such enzyme has not been isolated and studied in detail incacao and it is therefore still questionable, which cacao enzyme mightbe responsible for the generation of hydrophobic amino acids requiredfor cocoa flavor.

[0014] Though some aspects of cocoa flavor production have beenelucidated so far there is still a need in the art to fully understandthe processes involved, so that the manufacture of cocoa flavor mayeventually be optimized.

SUMMARY OF THE INVENTION

[0015] The present invention provides means for further elucidating theprocesses involved in the formation of cocoa-specific aroma precursorsduring the fermentation of cacao seeds, to improve the formation ofcocoa flavor during processing and manufacturing and eventuallyproviding means assisting in the artificial production of cocoa flavor.

[0016] This problem has been solved by providing a nucleotide sequenceencoding a novel carboxypeptidase from cacao beans (termed cacaoCP-III), which is identified by SEQ. ID. No. 1, or functionalderivatives thereof having a degree of homology that is greater than80%, preferably greater than 90% and more preferably greater than 95%.

[0017] It will be appreciated by the skilled person that a gene encodinga specific polypeptide may differ from a given sequence according to theWobble hypothesis, in that nucleotides are exchanged that do not lead toan alteration in the amino acid sequence. Yet, according to the presentinvention also nucleotide sequences shall be embraced, which exhibit anucleotide exchange leading to an alteration of the amino acid sequence,such that the functionality of the resulting polypeptide is notessentially disturbed.

[0018] This nucleotide sequence may be used to synthesise acorresponding polypeptide by means of recombinant gene technology, inparticular a polypeptide as identified by SEQ. ID. No. 2.

[0019] As has been shown in a comparison with other carboxypeptidasesfrom other plants the present enzyme does not show a substantialhomology to any of the carboxypeptidases known so far. Since it isassumed, that cocoa may furthermore contain additional carboxypeptidasesthat might exhibit a higher homology to the carboxypeptidases known sofar it must be considered as a surprising fact that this very enzyme hasbeen detected.

[0020] For producing the polypeptide by recombinant means, thenucleotide of the present invention is included in an expression vectordownstream of a suitable promoter and is subsequently incorporated intoa suitable cell, which may be cultured to yield the polypeptide ofinterest. Suitable cells for expressing the present polypeptide includebacterial cells, such as e.g. E. coli, or yeast, insect, mammalian orplant cells.

[0021] The present DNA sequence may also be incorporated directly intothe genome of the corresponding cell by techniques well known in theart, such as e.g. homologous recombination. Proceeding accordingly willprovide a higher stability of the system and may include integration ofa number of said DNA-sequences into a cell's genome.

[0022] The cells thus obtained may in consequence be utilized to producethe polypeptide in batch culture or using continuous procedures, withthe resulting polypeptide being isolated according to conventionalmethods.

[0023] The recombinant carboxypeptidase obtained may be used for themanufacture of cocoa flavor. To this end, the enzyme described hereinmay be utilized in an artificial trial run, wherein a mixture ofdifferent proteins, such as cacao storage proteins, or proteinhydrolysates of other resources, are subjected to enzymatic degradationby means of enzymes, known to be involved in proteolytic degradation toeventually assist in the production of flavor precursors. The enzyme maylikewise also be utilized in the production of cocoa liquor, and in themanufacture of chocolate.

[0024] Yet, the present invention also provides plants, in particularcacao plants, comprising a recombinant cell, containing one or moreadditional copies of the carboxypeptidase of the present invention. Sucha cacao plant will produce beans, which will exhibit a modifieddegradation of storage proteins when subjected to the fermentationprocess, allowing a more rapid degradation or a pattern of hydrolysisthat yields a higher level of cocoa flavor precursor, since a higheramount of carboxypeptidase will be present.

[0025] The carboxypeptidase of the present invention may also be used toproduce other transgenic plants such as soybean and rice, producingseeds with this new protein modifying enzyme.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0026] In the figures,

[0027]FIG. 1 shows a scheme illustrating a potential process for theproteolytic formation of cocoa-specific aroma;

[0028]FIG. 2 shows the cloning strategy used for the isolation of a cDNAencoding a carboxypeptidase from Theobroma cacao;

[0029]FIG. 3 shows a comparison of the hydrophilicityPlot-Kyte-Doolittle for the cacao CP-III sequence with Barley CP-MI,CP-MII and CP-MIII;

[0030]FIG. 4 shows a Northern blot analysis of cacao CP-III.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] As described above, it was suggested that a carboxypeptidasecould be involved in the production of cocoa flavor precursors duringcacao fermentation. However it was not known in the art which cacaocarboxypeptidase carried out this function considering that five classesof carboxypeptidases (Type I-V) have been identified in different plantsby references to differences in substrate specificities, molecularweights and chromatographic profiles. Furthermore 50 sequences havinghomologies with serine carboxypeptidases exist in the completedArabidopsis genome.

[0032] The following examples illustrate the invention further withoutlimiting it thereto. In the examples the following abbreviations havebeen used:

[0033] PCR: Polymerase Chain Reaction

[0034] RACE: Rapid Amplification cDNA Ends

[0035] cDNA: complementary deoxyribonucleic acid

[0036] mRNA: messenger ribonucleic acid

[0037] DEPC: Diethyl pyrocarbonate

[0038] 3,4-DCI: 3,4-dichloroisocoumarin

EXAMPLES

[0039] Materials

[0040] Cacao (Theobroma cacao L.) seeds (male parent unknown) from ripepods of clone ICS 95 were provided by Nestle ex-R&D Center Quito(Ecuador). The seeds were taken from the pods immediately after arrivalat Nestle Research Center Tours (4-5 days after harvesting). The pulpand the seed coat were eliminated and the cotyledons were frozen inliquid nitrogen and stored at −80° C. until use.

[0041] Preparation of mRNA

[0042] Total RNA was prepared using the following method. Two seeds wereground in liquid nitrogen to a fine powder and extraction was directlyperformed with a lysis buffer containing 25 mM Tris HCl pH8, 25 mM EDTA,75 mM NaCl, 1% SDS and 1M P-mercaptoethanol. RNA was extracted with onevolume of phenol/chloroform/isoamylalcohol (25/24/1) and centrifuged at8000 rpm, 10 min at 4° C. The aqueous phase was extracted a second timewith one volume of phenol/chloroform/isoamylalcohol (25/24/1). RNA wasprecipitated with 2M lithium chloride at 4° C. overnight. The RNA pelletobtained after centrifugation was resuspended in DEPC treated water anda second precipitation with 3M sodium acetate pH 5.2 was performed inpresence of two volumes of ethanol. The RNA pellet was washed with 70%ethanol and resuspended in DEPC treated water. Total RNA was furtherpurified using the Rneasy Mini kit from Qiagen®.

[0043] Cloning of a carboxypeptidase cDNA

[0044] Cloning Strategy

[0045] A 1.5 kb 5′ end fragment of a carboxypeptidase from cacao seedwas amplified by RT-PCR using a degenerate oligonucleotide. Based on thesequence of this fragment, a primer was designed to amplify a 3′-endfragment. Finally, a full-length cDNA (cacao CP-III) was amplified usingprimers specific to both extremities (FIG. 2).

[0046] Primer Design

[0047] A search for carboxypeptidase sequences in the GenBank databaselead to the identification of several plant sequences. A multiplealignment of these sequences revealed the presence of conserved regions.The conserved sequence MVPMDQP located near the histidine catalytic sitehas been used to design a degenerate oligonucleotide in the antisenseorientation: pCP2r (5′-GGYTGRTCCATNGGNACCAT) (SEQ ID No. 3).

[0048] Synthesis of cDNA

[0049] Total RNA (see above) was used to synthesise first strand 3′ and5′ cDNAs with the SMART RACE cDNA Amplification Kit (Clontech, USA).Synthesis has been performed exactly as described in the kitinstructions using 1 μg of total RNA and the Superscript™ MMLV reversetranscriptase (Gibco BRL, USA). After synthesis, cDNAs were useddirectly for PCR or kept at −20° C.

[0050] 5′ RACE Amplification

[0051] Specific cDNA amplification was performed with 2.5 μl of thefirst strand 5′ cDNA in 50 μl buffer containing: 40 mM Tricine-KOH pH8.7, 15 mM KOAc, 3.5 mMMg(OAc)₂, 3.75 μg/ml BSA, 0.005% Tween-20, 0.005%Noninet-P40, 0.2 mM dNTP's, 14 pmoles of pCP2r primer, 5 μl of 1OXUniversal primer Mix (UPM) and 1 μl 5OX Advantage 2 polymerase Mix(Clontech, USA). Amplification was performed in a Bio-med thermocycler60 (B. Braun). A first denaturation step (94° C., 2 min) was followed by35 cycles of denaturation (94° C., 1 min), primer annealing (55° C., 1.5min) and extension (72° C., 2 min). The extension time was increased by3 sec at each cycle. Amplification was ended by a final extension step(72° C., 10 min). The amplified fragment was cloned in pGEMO-T vectorand sequenced.

[0052] 3′ RACE PCR

[0053] The sequence information obtained after the sequencing of the 5′end fragment was used to design a specific oligonucleotide pCP5(5′-GCTTTTGCTGCCCGAGTCCACC) (SEQ ID No. 4), which was used for 3′-RACEamplification. 3′-RACE PCR was performed with 2.5 gl of SMART singlestrand 3′ cDNA in 50 μl buffer containing 40 mM Tricine-KOH pH 8.7, 15mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA, 0.005% Tween-20, 0.005%Nonidet-P40, 0.2 mM dNTP's, 10 pmoles of pCP5 primer, ¹⁰¹t of 1OXUniversal primer Mix (UPM) and 1 μl 5OX Advantage 2 polymerase Mix(Clontech, USA). Amplification was performed via touchdown PCR, in aBio-med thermocycler 60 (B. Braun).

[0054] A first denaturation step (94° C., 1 min) was followed by:

[0055] 5 cycles including denaturation at 94° C. for 30 sec andannealing/extension at 72° C. for 3 mm

[0056] 5 cycles including denaturation at 94° C. for 30 sec andannealing/extension at 70° C. for 30 sec and 72° C. for 3 min

[0057] 30 cycles including denaturation at 94° C. for 30 sec andannealing/extension at 68° C. for 30 sec and 72° C. for 3 min.

[0058] The amplified fragment was cloned in PGEMO-T vector andsequenced.

[0059] Full Length cDNA

[0060] The sequence information obtained after the sequencing of 5′-and3′-RACE fragments was used to design two specific oligonucleotides.

[0061] pCP8: A sense primer (5′-CAAAGAGAAAAAGAAAAGATGGC) (SEQ ID No. 5)

[0062] pCP7r: A reverse primer (5′-CCCCAGAGCTTTACGATACGG) (SEQ ID No.6).

[0063] PCR reaction was performed with 2.5 gl first strand cDNA in 50 μlbuffer containing: 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mMMg(OAc)₂, 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mMdNTP's, 10 pmoles of pCP8 primer, 10 pmoles of pCP7r primer and 1 gl 5OXAdvantage 2 polymerase Mix (Clontech, USA). Amplification was performedin a Bio-med thermocycler 60 (B. Braun). A first denaturation step (94°C., 1 min) was followed by 35 cycles of denaturation (94° C., 30 sec),primer annealing (63° C., 1 min) and extension (72° C., 2 min). Theextension time was increased by 3 see at each cycle. Amplification wasended by a final extension step (72° C., 10 min). The amplified fragmentwas cloned in pGEMO-T Easy vector and sequenced.

[0064] Sequencing and Analysis of DNA Sequences

[0065] cDNA sequencing has been performed by Eurogentech (Belgium) andESGS (France). Sequence analysis and comparison were performed withLion's software bioScout, Lasergene software (DNAStar) and Genedocprogramme.

[0066] The cacao CP-III cDNA sequence is 1768 bp long. A putativeinitiation start codon was assigned by comparison with othercarboxypeptidase sequences. It is located 25 bp from the 5′ end. Theopen reading frame is broken by a stop codon (TGA) at position 1549,followed by a putative polyadenylation signal (TATAAA) at position 1725.

[0067] Cacao CP-III encodes a 508 amino acid type III carboxypeptidase Cwith a predicted molecular weight of 56 kDa and a μl of 5.04. Thecatalytic amino acids are present at position Ser², Asp and His⁴⁷³.Hydrophilicity analysis (FIG. 3) reveals that cacao CP-III encodes ahydrophilic protein with a very hydrophobic N-terminal end, indicatingthe presence of a signal peptide.

[0068] Northern Blot Analysis

[0069] Total RNA samples were separated on 1.5% agarose gel containing6% formaldehyde (FIG. 4). After electrophoresis, RNA was blotted ontonylon membranes (Appligene) and hybridized with ³²P-labeled cacao CP-IIIprobe at 65° C. in 250 mM Na-phosphate buffer pH 7.2, 6.6% SDS, 1 mMEDTA and 1% BSA. Cacao CP-III cDNA fragment was amplified by PCR usingpCP8 and pCP7R primers and labelled by the random priming procedure(rediprime II, Amersham Pharmacia Biotech). Membranes were washed threetimes at 65° C. for 30 min in 2×SSC, 0.1% SDS, in 1×SSC, 0.1% SDS and in0.5×SSC, 0.1% SDS.

1 6 1 1768 DNA Theobroma cacao 1 gactctcaaa gagaaaaaga aaagatggcaaatccgaaaa tcttataccc gttttctgtt 60 tcccttctct tcctcatttc catctcctccgcggccgctt cctccttctt agacgagcgg 120 cgactcggag gatcaagttt cccctcgatacatgcgaaga agttgataag ggagttgaat 180 ttgtttccta aggaggaagt caacgtcgttgatggaggcc aggtttcctt accggaggat 240 tcgaggttgg tggagaagcg gttcaagttcccgaatttgg cggtgcctgg tggggtttcc 300 gttgaggatt tgggtcatca tgctggttattacaagctag ctaattctca tgatgccaga 360 atgttctatt tcttctttga atcacgaaatagcaaaaagg accctgttgt aatctggttg 420 actggagggc cagggtgtag tagtgaattggctttgtttt atgaaaatgg tccttttacc 480 attgctgaga acatgtctct tatttggaatcagtatggtt gggacatggc atcaaacctt 540 ctgtatgtgg accaacccat tggtaccggctttagttata gttctgatag aagggacatt 600 cgtcataatg aagatgaagt tagcaacgacctatatgact tcttacaggc attctttgct 660 gaacaccctg agtttgaaaa gaatgacttttatataactg gagaatcata tgctgggcac 720 tacattccag cttttgctgc ccgagtccaccaaggaaaca aagctaaaga tggaattcat 780 ataaacctaa agggatttgc tattggtaatggcctgactg accctgcaat ccagtataaa 840 gcttacacag attatgcttt ggacatgggggtaattaaga agtctgacta caatcgtatc 900 aacaagctgg ttccagtttg tgaaatggcaataaagcttt gtggcactga tggcacaatc 960 tcttgcatgg cttcatattt tgtctgcaatgccatattca ctggcatcat ggcacttgct 1020 ggcgatacaa attactacga cattagaacgaaatgtgaag ggagcctttg ctatgacttc 1080 tcaaacatgg agacatttct gaaccaggaatctgttaggg atgcccttgg agttgggagt 1140 attgactttg tgtcctgcag tcctacagtgtatcaggcca tgctggttga ctggatgagg 1200 aatcttgaag ttggcattcc tgctctccttgaggatggtg tcaagcttct tgtatatgct 1260 ggagaatatg atctcatctg caactggcttggcaattcga gatgggtcca tgcaatggaa 1320 tggtctggtc agaaggagtt tgtagcatctcctgaggttc cttttgtcgt tgatggctca 1380 gaagcaggag tcttgagaac tcatggacctcttggtttcc taaaggttca cgatgcaggt 1440 cacatggttc ctatggacca gccaaaggcagcattggaga tgctgaagcg gtggactaag 1500 ggtacattat ctgaagctgc cgattcagagaaattggttg ctgaaatatg atttccatca 1560 ttgcactgct tgcatacaat ttagttggcattagaatggg aatagccgta tcgtaaagct 1620 ctggggtttc tatgtatgcc tgtaaataattgcatgttaa tgctagtaca atggtatctt 1680 tgttttttga agatcaccta ctgaacttatatgaatcaag gacttataaa aatcttctaa 1740 aaaaaaaaaa aaaaaaaaaa aaaaaaaa1768 2 508 PRT Theobroma cacao 2 Met Ala Asn Pro Lys Ile Leu Tyr Pro PheSer Val Ser Leu Leu Phe 1 5 10 15 Leu Ile Ser Ile Ser Ser Ala Ala AlaSer Ser Phe Leu Asp Glu Arg 20 25 30 Arg Leu Gly Gly Ser Ser Phe Pro SerIle His Ala Lys Lys Leu Ile 35 40 45 Arg Glu Leu Asn Leu Phe Pro Lys GluGlu Val Asn Val Val Asp Gly 50 55 60 Gly Gln Val Ser Leu Pro Glu Asp SerArg Leu Val Glu Lys Arg Phe 65 70 75 80 Lys Phe Pro Asn Leu Ala Val ProGly Gly Val Ser Val Glu Asp Leu 85 90 95 Gly His His Ala Gly Tyr Tyr LysLeu Ala Asn Ser His Asp Ala Arg 100 105 110 Met Phe Tyr Phe Phe Phe GluSer Arg Asn Ser Lys Lys Asp Pro Val 115 120 125 Val Ile Trp Leu Thr GlyGly Pro Gly Cys Ser Ser Glu Leu Ala Leu 130 135 140 Phe Tyr Glu Asn GlyPro Phe Thr Ile Ala Glu Asn Met Ser Leu Ile 145 150 155 160 Trp Asn GlnTyr Gly Trp Asp Met Ala Ser Asn Leu Leu Tyr Val Asp 165 170 175 Gln ProIle Gly Thr Gly Phe Ser Tyr Ser Ser Asp Arg Arg Asp Ile 180 185 190 ArgHis Asn Glu Asp Glu Val Ser Asn Asp Leu Tyr Asp Phe Leu Gln 195 200 205Ala Phe Phe Ala Glu His Pro Glu Phe Glu Lys Asn Asp Phe Tyr Ile 210 215220 Thr Gly Glu Ser Tyr Ala Gly His Tyr Ile Pro Ala Phe Ala Ala Arg 225230 235 240 Val His Gln Gly Asn Lys Ala Lys Asp Gly Ile His Ile Asn LeuLys 245 250 255 Gly Phe Ala Ile Gly Asn Gly Leu Thr Asp Pro Ala Ile GlnTyr Lys 260 265 270 Ala Tyr Thr Asp Tyr Ala Leu Asp Met Gly Val Ile LysLys Ser Asp 275 280 285 Tyr Asn Arg Ile Asn Lys Leu Val Pro Val Cys GluMet Ala Ile Lys 290 295 300 Leu Cys Gly Thr Asp Gly Thr Ile Ser Cys MetAla Ser Tyr Phe Val 305 310 315 320 Cys Asn Ala Ile Phe Thr Gly Ile MetAla Leu Ala Gly Asp Thr Asn 325 330 335 Tyr Tyr Asp Ile Arg Thr Lys CysGlu Gly Ser Leu Cys Tyr Asp Phe 340 345 350 Ser Asn Met Glu Thr Phe LeuAsn Gln Glu Ser Val Arg Asp Ala Leu 355 360 365 Gly Val Gly Ser Ile AspPhe Val Ser Cys Ser Pro Thr Val Tyr Gln 370 375 380 Ala Met Leu Val AspTrp Met Arg Asn Leu Glu Val Gly Ile Pro Ala 385 390 395 400 Leu Leu GluAsp Gly Val Lys Leu Leu Val Tyr Ala Gly Glu Tyr Asp 405 410 415 Leu IleCys Asn Trp Leu Gly Asn Ser Arg Trp Val His Ala Met Glu 420 425 430 TrpSer Gly Gln Lys Glu Phe Val Ala Ser Pro Glu Val Pro Phe Val 435 440 445Val Asp Gly Ser Glu Ala Gly Val Leu Arg Thr His Gly Pro Leu Gly 450 455460 Phe Leu Lys Val His Asp Ala Gly His Met Val Pro Met Asp Gln Pro 465470 475 480 Lys Ala Ala Leu Glu Met Leu Lys Arg Trp Thr Lys Gly Thr LeuSer 485 490 495 Gln Ala Ala Asp Ser Glu Lys Leu Val Ala Glu Ile 500 5053 20 DNA Artificial Sequence Description of Artificial Sequence Primer 3ggytgrtcca tnggnaccat 20 4 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 gcttttgctg cccgagtcca cc 22 5 23 DNAArtificial Sequence Description of Artificial Sequence Primer 5caaagagaaa aagaaaagat ggc 23 6 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 6 ccccagagct ttacgatacg g 21

What is claimed is:
 1. A nucleotide sequence coding for acarboxypeptidase and having a sequence as identified by SEQ. ID. No 1 orfunctional variants thereof having a degree of homology of more than90%.
 2. A polypeptide encoded by the nucleotide sequence of claim
 1. 3.The polypeptide of claim 2, which is identified by SEQ. ID. No
 2. 4. Avector containing the nucleotide sequence of claim
 1. 5. A cellcontaining the nucleotide sequence of claim
 1. 6. A bacterial, a yeast,an insect, a mammalian or a plant cell containing the nucleotidesequence of claim
 1. 7. A cacao cell containing the nucleotide sequenceof claim
 1. 8. A transgenic plant containing a cell of claim
 7. 9. Amethod for synthesizing a carboxypeptidase which comprises obtaining thenucleotide sequence of claim 1 and utilizing the nucleotide sequence tosynthesize the carboxypeptidase.
 10. A method for manufacturing a cocoaflavor which comprises subjecting the polypeptide of claim 2 to anenzymatic degradation to obtain cocoa flavor precursors formanufacturing the cocoa flavor.
 11. A method for manufacturing a cocoaliquor which comprises subjecting the polypeptide of claim 2 to anenzymatic degradation to obtain cocoa flavor precursors formanufacturing the cocoa liquor.
 12. A method for manufacturing achocolate which comprises subjecting the polypeptide of claim 2 to anenzymatic degradation to obtain cocoa flavor precursors formanufacturing the chocolate.
 13. A method for hydrolysing proteins whichcomprises obtaining a polypeptide of claim 2 and utilizing thepolypeptide to hydrolyse the proteins.
 14. The method of claim 13wherein the proteins are derived from food material.
 15. A process forproducing a cocoa flavor comprising subjecting the polypeptide of claim2 to an enzymatic degradation to obtain cocoa flavor precursors formanufacturing the cocoa flavor.
 16. A cocoa flavor produced by themethod of claim
 10. 17. A compositions comprising a food or beverageproduct and a flavor effective amount of the cocoa flavor of claim 16.